Our long term goal is to characterize the cellular and molecular mechanisms regulated by glutamate transport activity under both normal and pathophysiological conditions. Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system, and as such plays a key role in neurological diseases involving hyperexcitability and excitotoxic cell death. Glutamate transport maintains extracellular glutamate concentrations below neurotoxic levels and loss of transporter protein is associated with several neurodegenerative disorders. We have tested the hypothesis that glutamate transporters play a key role in preventing cell death in limbic seizures, by generating a transgenic mouse model of astrocytic EAAT2 (Glt-1) overexpression. The EAAT2 protein is tagged with GFP (green fluorescent protein) and expression is driven by the astrocyte-specific GFAP promoter. Overexpression of the EAAT2 transgenic protein results in a 2-3 fold increase in hippocampal and cerebrocortical synaptosomal D-aspartate uptake. In a kainic acid (KA) model of temporal lobe epilepsy, increased glutamate transport results in an 80 percent decrease in hippocampal cell death compared to the level of cell death following KA-induced seizures in a wild-type age-matched animals. Surprisingly, we also found that increased glutamate uptake in the EAAT2 transgenic blunted network excitability and immediate early gene responses. These data, taken together with recent findings from other investigators, indicate that in addition to maintaining low steady-state concentrations of glutamate around the synaptic cleft, transporters may mediate a more rapid control of synaptic efficacy. Since glutamate is the major excitatory neurotransmitter in the CNS, we hypothesize that increased glutamate transport will blunt network excitability in most, if not all, CNS models of seizure-related hyperexcitability. To test this central hypothesis, we will use three models of status epilepticus (KA, pilocarpine and kindling). To extend our preliminary studies we will examine the acute actions of the glutamate analogue kainic acid using two in vitro slice preparations (hippocampal and piriform cortex) to determine if increased glutamate transport alters the threshold, frequency or duration of epileptiform activity. We will also inject KA, NMDA or AMPA into the hippocampus and record EEG activity to elucidate differences in seizure onset, frequency and duration in the presence of increased glutamate uptake (Specific Aim 1). To determine if the effects of increased glutamate transporter expression are generalized or limited to convulsants acting directly through a glutamate receptor pathway, we will use pilocarpine, that acts through muscarinic receptors, to generate seizure activity in vivo and in an in vitro hippocampal slice preparation to compare wild-type to EAAT2 transgenic responses (Specific Aim 2). Finally we will use two kindling models of status epilepticus to determine if increased glutamate transport activity lowers the threshold, rate of kindling acquisition, molecular plasticity or degree of cell death in PAAT2 transgenic mice compared with age-matched wild-type animals Specific Aim 3).